Principles of mass spectrometry

A mass spectrometer consists of three components: an ion source, which converts the sample into gas phase ions, a mass analyzer which separates the ions according to their mass-to-charge ratio, and finally a detector, which calculates the abundances of each ion present. The data generated are displayed as a spectrum of the relative abundance of ions as a function of their mass-to-charge ratio (m/z).

Prior to MS analysis, the C3 protein was reduced in order for the α and β chains to be separated and analysed separately. The two chains were separated by SDS-PAGE and then fragmented further following enzyme digestion with trypsin, which cleaves proteins on the C-terminal side of lysine and arginine residues, unless they are immediately followed by proline. As a consequence, the treatment of proteins with trypsin generates peptides with an average length

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of approximately ten to fifteen amino acid residues which is well suited for MS detection.

Following fragmentation of the protein, the peptide fragments are subjected to tandem MS analysis (MS/MS). The masses of the sample fragments are determined in a MS survey scan after which peptide ions of interest are isolated according to their mass-to-charge ratio value. Further fragmentation of the selected peptide ion species occurs by the addition of an inert gas such as Argon that imparts internal energy to the ions as they collide. The fragmented ions pass through the mass analyser which determines the m/z values. The molecular mass of amino acids that have been affected by PTM is altered and can be detected by increments or deficits in mass on the MS/MS spectra.

This technique very useful for studying changes as a result of PTM as it is highly sensitive, and requires very little sample. It is able to detect changes in molecular mass that correspond to modifications at both the peptide and protein level and can therefore identify the type and location of the PTM.

This technique does however have limitations as the processes of digestion, ionization and MS/MS activation can lead to dissociation of the PTM from the protein. This tends to occur if the covalent bond between the amino acid and the PTM is chemically unstable and so the PTM may not be detected. An added complication is that the presence of PTM may affect the efficiency of trypsin in cleaving the protein, leading to larger peptide fragments that are less suitable for analysis.

2.12.1 Gel processing and tryptic digestion

25 µL of each sample was buffer exchanged into 50 mM ammonium bicarbonate using Vivaspin 500 3000 kDa MWCO centrifugal concentrators

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(Sartorius Stedim). 3 x 25 µL washes with ammonium acetate were performed. Samples were then reduced by addition of DTT to 5 mM with heating to 56 °C for 20 min and then alkylated by addition of iodoacetamide to 15 mM with incubation in the dark at room temperature for 15 min. Trypsin in 50 mM ammonium acetate was then added to the samples in a protein:protease ration of 1:50 w/w and incubated for 18 hours at 37 °C with shaking. After briefly vortexing and centrifuging, the trypsin reaction was quenched by addition of 5 µL of formic acid. Digested samples were stored at -80 °C until LC-MS analysis.

2.12.2 Liquid chromatography-mass spectrometry

Liquid chromatography-mass spectrometry (LC-MS/MS) analysis of the peptide mixtures was performed on an Ultimate 3000 nano LC system (Dionex, Amsterdam, Netherlands). 1 µl of each sample was loaded onto a C18 guard column and washed with 2% acetonitrile/0.1% formic acid for 5 min at 25 µl/min. After valve switching, the peptides were then separated on a PepMap C18, 100µm i.d.x15 cm analytical column (Dionex, Amsterdam, NL) by gradient elution of 2-60% solvent B (0.05% formic acid in 20% water/80% acetonitrile) in solvent A (0.05% formic acid in 98% water/2% acetonitrile) over 60 minutes at 0.35 µl/min. The column eluant was directly interfaced to a quadrupole-ion mobility-orthogonal time of flight mass spectrometer (Synapt G2S, Waters UK, Manchester) via a Z-spray nanoflow electrospray source. The MS was operated in positive TOF mode using a capillary voltage of 3.8 kV, cone voltage of 20 V, backing pressure of 7.91 mbar and a trap bias of 4.1 V. The source temperature was 80 °C. Argon was used as the buffer gas at a pressure of 9.1×10-3 mbar in the trap and transfer regions. Mass calibration was performed by a separate injection of sodium iodide at a concentration of 2 µg/µl. [Glu1]-Fibrinopeptide B

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at 2 µM was infused as a lock mass calibrant with a one second lock spray scan taken every 30 seconds during acquisition. Ten scans were averaged to determine the lock mass correction factor. Data acquisition was using data dependent analysis with a one second MS over an m/z of 350-2000 being followed by three 1 second MS/MS taken of the three most intense ions in the MS spectrum above a threshold of 1000 counts per second. Collision energy applied was dependent upon charge state and mass of the ion selected. Dynamic exclusion of 60 seconds was used. Data processing was performed using the MassLynx v4.1 suite of software supplied with the mass spectrometer. Peptide MS/MS data were processed with ProteinLynx Global Server (Waters) and searched against UniProtKB/SwissProt database (release 2011/12). Search was limited to Homo sapiens species. Precursor mass accuracy was 10 ppm and fragments ion tolerance was 0.1 Da. Trypsin was specified as the protease with up to two missed cleavages. Carbamidomethylation was used as a fixed modification and variable modifiactions were specified as acetylation, glycation, deamidation and oxidation.